Disorders of keratinization (DOK) are severe, rare genetic skin disorders in which the central barrier function of the skin is disrupted and a compensatory pathway of hyper-proliferation is activated in either a localized area or throughout the entire skin surface. This results in a grossly compromised epidermis that fails to adequately protect against bacterial, chemical, and mechanical assault or to prevent transepidermal water loss. In early life, the consequences of these disorders can be life-threatening, with increased susceptibility to infection due to blistering and fissures, dramatically increased metabolic demands due to evaporative heat loss and increased epidermal turnover rate, and various associated comorbid conditions and systemic abnormalities which can persist throughout life. Prior studies have identified over 70 diverse genes for these disorders, yet clinical experience and our data show that these genes explain only a portion of heritability for DOK, which demonstrate marked locus and phenotypic heterogeneity, with greater than 20% of subjects without mutation in known genes. Next generation sequencing has provided a new opportunity to identify genes for DOK, and we have applied it successfully to identify several novel genetic causes of these disorders. We now propose to expand our large cohort of well-phenotyped DOK kindreds, to screen for mutations in common causative genes, and to employ exome sequencing in those subjects without mutation in common DOK genes to discover novel genetic causes of these disorders. We will employ patient-derived cells and tissue to interrogate the function of identified novel genes, and will employ transgenic skin equivalents to study and prove pathogenesis of identified mutations. For a limited number of novel genes not previously implicated in DOK, we will generate mouse models using CRISPR technology to further examine the effect of identified mutations on skin development and physiology. These studies will continue to identify molecular pathways central to the complex processes of epidermal differentiation and self-renewal, and will provide critical context for more detailed future biologc studies.